Method and apparatus for detection of a controlled substance

ABSTRACT

Techniques and devices for detecting and analyzing controlled substances and the like are discussed including highly reactive sensor molecules which are coated on a spectroscopic sample surface ( 4 ) and which may chemically react with a given analyte to form a covalently bonded adduct with spectral characteristics unique to the new adduct. The techniques provide the basis of a detection system with high sensitivity and high specificity in which the surface can even be washed to remove interfering or nonreactive compounds. The sensor molecules which comprise the coating ( 8 ) may have three major components: a central molecular scaffold (“CMS”), a “tether” terminated by a surface attachment group “SAG,” and a reactive functional group “RFG” which may be highly reactive towards certain classes of molecules. One or more modifiers or modifier groups “Z” which may serve to increase or decrease the reactivity of the RFG towards target analytes, or to modify the spectral characteristics of the adduct may also be included. Some sensor molecules include diazonium compounds, activated acyls, and nitrosos.

This application is the U.S. National Stage of International ApplicationNo. PCT/US98/12974, filed Jun. 24, 1998, which claims the benefit ofU.S. Provisional Application No. 60/050,716, filed Jun. 24, 1997, eachhereby incorporated by reference.

I. TECHNICAL FIELD

Generally this invention relates to the field of detecting the use ofcontrolled substances such as illicit drugs and the like. Morespecifically, the invention involves the field of Raman spectroscopy toaccomplishing detection and the sub-field known as Surface EnhancedRaman Scattering. In a less focused sense, this invention relates to amethod of detecting substances through the use of a coated surface andspectroscopic techniques. The invention also covers the use of a newcoating with a tethered reactive species that chemically binds to ananalyte. Through this new technique, the analyte is thenspectroscopically analyzed as part of a new chemical species which isthe reaction product of the analyte and the reactive species.

II. BACKGROUND ART

The field of sensing controlled substances is an area which has evolvedprimarily for the public good. As practically everyone knows, drug andalcohol abuse are significant problems for society. In fact, in 1998 theUnited Nations conducted an international anti-drug conference involvingover one hundred nations which are facing this societal challenge. Associety attempts to address this problem it has turned to increasinglysophisticated technical analysis to counter the abuser's attempt to hideeither the controlled substance or its use. Naturally, the principles ofanalytical chemistry have been relied upon for their ability to not onlydetect but to discriminate the presence of a controlled substance inminute amounts. Unfortunately at its present state, the field ofdetecting controlled substances still has difficulty in both sensing anddiscriminating the existence of some substances as well as in avoidingfalse positive indications. This invention provides a solution thatgreatly expands the techniques and accuracy available for a variety ofsubstances. It also provides a framework under which practicaladvantages can now be achieved. These advantages range from theseemingly simple ability to provide a single sensor for a variety ofdrugs as well as the ability to now be able to discriminate betweencontrolled substances and some chemically similar uncontrolledsubstances. This latter aspect can be significant because in a varietyof applications such as Olympic drug testing and the like, it has becomedifficult to accurately sense and distinguish the difference betweencertain substances which are legally available for use and those whichare truly illegal substances. In a broader sense, the invention alsoprovides an expansion to the principles of analytical chemistry since itmay be applied in other areas as well.

The field of analytical chemistry dates back at least to Pliny the Elder(AD 23-79) who first described the use of an extract from gallnuts thatturns black in the presence of iron sulfate. This allowed him todetermine if copper sulfate was contaminated with iron sulfate. Thissimple concept of chemical analysis has grown into analytical chemistrywhich is one of the four disciplines of modern chemistry. Analyticalchemistry encompasses a variety of fields such as clinical chemistry,environmental chemistry, geochemistry, and forensic chemistry. Thetechniques of analytical chemistry have grown from the simple wetchemical analysis discovered by Pliny the Elder to very sophisticatedinstrumental methods. Early analytical chemistry relied on visualobservation of color changes or the precipitation of a compound toquantitate materials. This meant that the sensitivity was often limitedto the visual acuity of the chemist. Instruments have largely replacedthese visual techniques, since it is possible to electronically detectchanges in light intensity and wavelength with vastly superiorsensitivity.

The electronic detection of changes in light intensity and itsseparation into different wavelengths is the basis of the field ofanalytical spectroscopy. This is an area in which some type of analyte,namely, some substance or chemical which is desired to be studied, isexposed to some wavelength of energy. This wavelength may be a singularwavelength such as a laser often provides, or it may be many differentwavelengths. To provide the information desired, the analyte then causessome type of change in that incident energy and thus results in sometype of change in intensity of at least one wavelength of energy whichis characteristic of the analyte. Thus the wavelength of energy to whichthe analyte is exposed and the changed signal resulting usually vary.Naturally, the incident energy may be present in a variety of forms asthose aware of the wave-particle duality may easily understand.Essentially, however, all that spectroscopy involves is an incidentwavelength which is somehow affected by an analyte to result in achanged signal. This signal may be a singular wavelength or may be abroad spectrum of wavelengths of emission or adsorption. Thus,“spectroscopy” as intended here is not intended to be limited to onlysome type of slit-based instrument, but rather is intended to fullyencompass the areas of analytical chemistry in which changes inwavelengths of energy are studied to gain information with regards to ananalyte. Conversely, it should be understood that other fields or areasof study which do not involve changed wavelengths have not been viewedas particularly relevant to this field. For example, the areas ofchromatography and the like which act to separate substances,immunoassays which transiently bind substances for nonspectroscopicpurposes, and the like, have not been viewed as particularly relevant tothe fields in which this invention relates.

As mentioned earlier, the field of sensing controlled substances faces avariety of limitations. These range from imperfect discrimination (suchas in the Olympic drug testing scenarios) to practical challenges suchas the need to have different tests for different substances. Whenconsidering spectroscopic techniques, great improvement has occurredthrough the introduction of a technique known as Raman spectroscopy.Raman spectroscopy was discovered by Sir Chandrasekhara Venkata Raman inthe early 1900s who found that different chemicals sometimes causedunique scattering of an incident wavelength of energy. Since thescattering was largely unique to each chemical, the analysis of thespecific scattering thus provided information from which specificchemical detection and identification could be achieved. Unfortunately,limits remained even with the introduction of Raman spectroscopy.

In 1976, a new spectroscopic technique was discovered that is sensitiveto interfaces. This technique has been coined Surface Enhanced RamanScattering (SERS). SERS tends to give large enhancements of Ramanscattering in the presence of certain prepared metallic surfaces. TheSERS technique has been applied to a variety of problems—not only thoseassociated with analytical chemistry—and more recently has been thesubject of several publications and patents for analytical chemistry.This technique generally involved some type of attachment of an analyteto a metal surface or to a coating on a metal surface such as gold orsilver.

Even in the broader area of general analytical chemistry, initially, thecoatings were not tethered to the metal surface. In U.S. Pat. No.5,326,211 relating to analytical chemistry in general, Carron and Mullenshowed that it was possible to coat a surface with a dye that hadcomplexed with a metal ion to serve as a metal ion detector. Again inthe broader field of analytical chemistry, Angel was awarded an earlypatent, U.S. Pat. No. 4,781,458 for the determination of analytesadsorbed directly to metals surface or partitioned onto a coating. Evenmore recently some publications by Carron and U.S. Pat. No. 5,327,211disclosed the use of SERS with coatings that contain thiols to tetherthe coatings to a silver surface. That disclosure specifically addressesthe use of SERS coatings on a fiber optic to allow for remote sensing.Carron, et. al, has demonstrated that the coatings mimic separationscience coatings and also serve to stabilize the SERS substrate to giveit longevity. The coatings can also provide an internal standard thatallows one to use relative intensities to determine a calibration thatcan be used to find the concentration of the analyte.

The general techniques of SERS has been well established and isdiscussed to some degree not only in the above general analyticalchemistry references, but also in a variety of references ranging fromtext books to additional patents such as U.S. Pat. No. 5,693,152 toCarron, U.S. Pat. No. 5,255,067 to Carrabba, and U.S. Pat. Nos.5,266,498, 5,376,556, and 5,567,628 to Tarcha in the immunoassay field.To the extent necessary, each of these references is hereby incorporatedby reference to provide additional understandings as they relate to theRaman and SERS techniques generally.

One of the perspectives that has evolved to those focusing on thesetechniques has been a perspective that suggests that it may be moreappropriate to avoid changing any structures or spectral characteristicsof an analyte during its spectroscopic analysis. This perspective seemsrooted in the understanding that, naturally, if one wants to detect theanalyte itself, it would be better not to alter the analyte orcontrolled substance. Thus, the coatings developed to date foranalytical applications of SERS have been coatings that weakly interactwith the analyte to produce a reversible measurement of the analyteconcentration. These include alkylthiols that mimic the nonpolarcoatings used in reverse phase HPLC, pH and metal sensitive coatingsthat mimic ion chromatography, and oxide coatings that mimic normalphase chromatography.

By weakly interacting with the analyte to produce a measurable spectrum,the goal of seeing only the analyte spectrum had been satisfied. As aresult, the techniques of Raman spectro-scopy and specifically that ofSurface Enhanced Raman Scattering, had to some degree been viewed aslimited since the surfaces typically used in SERS have been therelatively unreactive substances of gold and silver. Since thesesubstances can cause the desired weak interactions in only a fewsituations, these techniques, while powerful for certain analytes, hadnot been as greatly exploited as possible in the field of detection ofcontrolled substances. In essence, since a goal was that the surfaceitself created some sort of weak link with the controlled substance, andsince not many controlled substances would establish an appropriate linkwith the typically required gold or silver surfaces, these techniqueswere often not viewed as particularly appropriate to the field ofdetection and analysis of controlled substances or specific substancesand the like. The techniques were also viewed as somewhat limitedthemselves because it seemed that they could really only be used forthose specific types of analytes that happened to bond appropriately tothe required surface. Although eventually different sample surfaces didexist for certain different analytes, generally the selections were solimited that the techniques were perhaps underutilized.

One example of efforts to alter the surface involved in the SERStechnique is disclosed in U.S. Pat. No. 5,693,152 to Carron, one of thepresent inventors. In that patent, Carron disclosed a technique tomodify the surface enhanced Raman scattering (SERS) detector by applyinga stabilizing coating on the SERS surface. The coating applied wouldreproduce or mimic the specific separation procedure being utilized thusthis method could be used universally for all types of analytes andseparation methods. Similar to the invention disclosed in a patent byCarrabba et al (U.S. Pat. No. 5,255,067), a roughened surface substratewas used to improve SERS detection efficiency in gas chromatography.However, although the coating disclosed in '152 patent can locallyincrease the analyte concentration and improve linking affinity betweenthe coating and the analyte, the attachment of these analytes to thecoatings are still through weaker linking mechanisms, primarily by meansof adsorption or other weak forces.

Similarly, other, perhaps unrelated fields have seen efforts to alterbinding sites in the immunoassay area. As mentioned earlier, the use ofSERS technology for immunoassays has been disclosed by Tarcha et al. InU.S. Pat. Nos. 5,266,498, 5,376,556, and 5,567,628. In their disclosuresthe authors designed a Raman active reporter which is bound to aspecific binding member. However, this is not only an immunoassaymethod, it involves attachment of an analyte to a binding member throughweaker techniques and thus even though it is in an unrelated area, seemsto show the pervasiveness of the perspectives and attitudes of thoseinvolved in Raman spectroscopy.

In the focused field of detecting controlled substances, however, theproblems seem even more acute. Those involved in sensing controlledsubstances (and other, even uncontrolled substances) have faced problemswith the discrimination abilities between substances. The problems forprofessional athletes who may have taken some type cold medicine priorto their participation in the Olympic games has been highly popularized.From one perspective this problem may be viewed as a simple byproduct ofthe inability of the analytical techniques to adequately discriminatebetween a legal cold medicine and the remnants as a result of the use ofan illegal drug.

Another problem which is the fact that it is desirable to sensecontrolled substances in extremely low concentrations. Again, theseemingly irresolute requirement that the analyte or controlledsubstance only weakly binds with the spectroscopic surface has made itchallenging to sense such low concentrations because the equilibriumvalues are often such that the analyte would desorb from the surface andnot permit a sufficient build up to facilitate high quality detection.Further, a problem has existed in instances in which it was desired tonot only detect but to quantify the presence of either some controlledsubstance or more generally an analyte, over a broad ranges ofconcentrations. In those instances in which such spectroscopictechniques were found to apply, it was often the case that the rangesover which a specific sensor could be used made it practically difficultto achieve the technique.

Furthermore, because there are a great variety of controlled substances,one of the problems has been in the ability to use the specificspectroscopic technique to sense not only one specific type ofcontrolled substance but to sense a great variety of substances. Notonly have there existed limitations on which specific substancesinteract appropriately with which specific sensors, but there haveexisted seemingly fundamental limitations such that certain substancescould not be analyzed through Raman techniques. Naturally, while many ofthese problems are particularly acute in the focused field of sensingcontrolled substances, it can be easily understood that these problemsalso applied to more general applications of spectroscopic techniques aswell.

One way in which the apparent requirement of a weak interaction hasresulted in a difficulty is the practical fact that interferingsubstances are often present. While it would be desirable to removethese interfering substances, the weak interaction has made it moredifficult to remove the interfering substances without also removingsome analyte as well. Even the improved SERS coating disclosed in U.S.Pat. No. 5,693,152 highlights that this problem continued unsolved.Essentially, the problem was that while such a SERS coating couldspecifically adsorb material from the matrix, there were no ways ofwashing the surface to remove the interferences either unavoidably orpractically present in the matrix. Furthermore, since the interaction ofthe analyte with the surface was often viewed as necessarily reversible,it was implied that the interaction needed to be weak. Physically, thisoften meant that only a small amount of the analyte tended to interactwith the surface or could be detected in a given instance.

Thus, in the field of sensing controlled substances, there has been along felt but unsatisfied need for a detection technique which could beapplied to a great number of controlled substances and which could alsoprovide a higher degree of discrimination between such substancesespecially those which were not considered illegal. Even though thoseskilled in the field of sensing controlled substances appreciated thisdesire, they seemed not to have fully appreciated the nature of theproblem in that their perspective was driven by certain preconceptionswhich actually limited their ability to solve the problems with whichthey were faced. To some degree their substantial attempts failed tofill the need either because their field did not require the basicphysical understanding of the phenomenon or they simply assumed thatexisting techniques could not be adapted to their unique needs. For thisreason, it appears that those skilled in the art to some degree actuallytaught away from the direction in which the present inventors went.

As related to the broader field of analytical chemistry in general, itappears that similar perspectives also apply. For instance, while thosein the field of analytical chemistry in general well appreciated that itwas desirable to apply sensitive techniques such as Raman spectroscopyand the SERS techniques to a greater variety of substances, theirapparent tendency was to approach the problems from the perspective ofseeing primarily the analyte as opposed to some altered by-product.Again, while they had expended substantial efforts to expand theapplicability of the techniques, their focus toward physisorptionperhaps showed that they did not fully appreciate the nature of theproblem. They seemed thus to teach away from the direction in which thepresent inventors went pursuing avenues that in hindsight might beviewed as based on misperceptions to some degree. Not only were thesemisperceptions fostered by the initial desire to sense an unalteredanalyte, but they also were fostered by attitudes which seemed tosuggest that reversible and remote sensing arrangements were requiredfor some practical reason. Thus, to some degree the present inventionmight even be characterized as unexpected in the sense that it proposestechniques and substances which, prior to this invention, were not justdeemed suboptimal but rather were viewed as contrary to the goalstypically considered and the results typically desired.

In addition to the aspect of applying analytical techniques to a greatervariety of substances, those generally applying the techniques had longfelt a need for an ability to apply those techniques to greater rangesof concentrations and to greater varieties of chemicals during oneanalysis event. Even though a desire existed, they may not have fullyappreciated that the problem and solution lay not in sample preparationor non-spectroscopic techniques, but in adjustments to the analyticaltechnique itself or to the specific sensors involved in the analyticaltechnique. Efforts focussed in a direction different from those of thepresent inventors may have been due to the fact that those involved didnot to some degree fully appreciate that sensor chemistry could offerthe needed advances and simplifications.

III. DISCLOSURE OF THE INVENTION

To address these and other problems, the present invention involvestechniques and devices which offer improved spectroscopic analysiscapabilities. As applied to the field of sensing controlled substances,the invention involves the creation of a unique surface coating such asa diazonium or other type of coating which interacts with the substancein a wholly different manner. Rather than forming the weak interactionsthat were previously viewed as perhaps required, the invention creates awhole different type of interaction, namely a full reaction such as inthe formation of a covalent bond to produce an entirely differentspecies, termed here in adduct. Since in most instances the formation ofthis covalent bond is irreversible, the rate of loss of the controlledsubstance or other analyte from the surface will be approximately zero.As a result, washing or other steps to remove interference substancescan now be accomplished. Thus, as a result, the invention offers agreatly expanded Surface Enhanced Raman Scattering technique throughwhich coatings may be altered as appropriate for specific substances andthrough which expanded ranges and sensitivities can now be accomplished.Thus the detection of substances which are controlled, substances of amedical nature, or simply some new types of analytes are now possible.In its approach, the invention breaks with what may have beenpreconceptions and utilizes a substance, such as diazonium, which ishighly reactive with the controlled substance or other analyte at issue.Thus the diazonium and the like may interact to permanently bond orperhaps covalently bond with the analyte as opposed to the more typicalweak chemisorption or physisorption. Through this reaction the diazoniummay be viewed as an advantage rather than a hindrance in that an actualadduct is created and rather than studying the analyte in isolation theresulting adduct is studied.

In a more basic form, the invention includes a method of detectingmolecules by chemically binding them to a surface and then using asurface-sensitive detection method. This is a significant improvementover the existing method of attracting molecules to a surface throughweak forces. The new technique increases the sensitivity of detection byforming a strong bond between the analyte and the surface coating. Theequilibrium between surface analytes (e.g. detectable analytes) andsolution analytes (e.g. not detectable since not captured by thesurface) may be characterized by an equilibrium constant. In one sense,the equilibrium constant describes the number of species at the surfacerelative to solution. In another sense, it describes the rate at whichmolecules go on the surface relative to the rate at which they come off.The present invention describes a method of forcing the off rate to beessentially zero and to thereby greatly increase the number of specieson the surface (e.g. the detectable species). This is one form ofsensitivity improvement. A second method of improvement comes from theability to wash the surface after the analyte has bonded to the surface.In many cases, the sensitivity of an analytical method has been limiteddue to the presence of large interfering backgrounds. This backgroundarose from species in the solution with the analyte that gave rise to asignal that was similar to that of the analyte. These interferingspecies can now be removed with this invention since the analyte isbound to the surface and the interfering species can be easily washedoff without affecting the analyte. (This, of course may, not work forevery analyte possible.) A third method of improvement comes from theability to distinguish between different analyte types. Because theadduct formed through formation of a covalent bond is a new and uniquechemical species, it will likely have unique spectroscopiccharacteristics. Since different analytes will form different adducts,they may be distinguished from each other on the basis of the differingspectra, thus decreasing the likelihood of mistaken identification.

In the field of sensing controlled substances, it is thus an object ofthe invention to provide techniques and devices which may be applied toa greater variety of controlled substances and which may be applied toall substances with a higher degree of sensitivity, with greater abilityto discriminate, and even with the possibility of confirmational systemsto be automatically in place to avoid false indications. In keeping withthese objects, it is a goal of the invention to offer a system which iscontrolled to a lesser degree by the equilibrium constants of a reactionand which even offers situations where the equilibrium constant isessentially zero. Similarly, a goal is to provide a system in whichinterfering substances and the like can be removed without concern ofremoving or somehow reducing the signatures available as a result of thecontrolled substance itself. A further goal is for a system whichpermanently binds the controlled substance to the sensor surface andthus can be analyzed at any location or any time without a high degreeof concern of degradation of the signal.

A more broadly stated goal for the invention in the field of sensingcontrolled substances is that of providing techniques which can bedesigned for specific chemicals in specific situations. Thus a goal isto provide a system which can be optimized through chemical design foreither specific substances or for specific ranges and broader substancesin one analytical event.

An object as it relates to the broader field of analytical chemistry ingeneral, as well as in the field of detecting controlled substances, isto provide a system in which internal standards can permit themonitoring and compensation for alterations in the illumination sourceas well as to provide techniques in which coatings rather than thesurfaces themselves can be used in a variety of detection techniques.Thus, the invention could be used with a large variety of analyticaltechniques, known to those in the art. Optical techniques such as Raman,fluorescence, or absorption spectroscopy could be used for detection.Mechanical detection would also be possible with sensitive mass sensors.Raman scattering may prove to be one of the most desirable detectionmethods. Surface Enhanced Raman Scattering (SERS) provides largeenhancements in the Raman scattering from molecules near certain metalsurfaces. The invention thus involves methods of anchoring our moleculespecific probes to this type of surface. Moreover, while the inventioncontains a fairly high specificity for specific classes of molecules itmay not need to be truly specific to a single molecule. SERS, of course,is a truly molecular-specific detection method. This means that acomplex mixture of species in a reactive class of compounds could reactwith the surface and SERS could differentiate and quantitate between thevarious species.

The use of SERS also provides a substantial improvement over manytechniques through the use of an internal standard. The internalstandard furnished by this invention can be the portion of the surfacetether that is unchanged by the reaction of the reactive functionalgroup (RFG) with the analyte. This portion of the surface coating maygive rise to a SERS signal that can be monitored simultaneously withthat of the analyte. A simple ratio of the two signals or a moresophisticated multi variate analysis can be used to give a detectionmethod that is independent of source intensity fluctuations orvariations in the detection throughput. This creates the possibility formore simplified and less expensive instrument design as well as designfor rugged, maintenance free use.

IV. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows spectra of a prototype coating, 4-amino phenyldisulfide,and its reactivity toward phenols. A) A SERS spectrum of 4-aminophenyldisulfide. The disulfide bond has broken and the surface speciesis 4-amino thiophenylate. B) A SERS spectrum of the coating used inFigure A after treatment to make it into a diazonium salt. C) A SERSspectrum of the coating in Figure B after exposure to phenol. The Ramanfeatures around 1512 cm−1 are indicative of the azo functionality. Theshows that the coating reacted with phenol to make an azo compound.

FIG. 2 is an example of the components that might be present in theinstrument required to read the results of the current invention. Theinput may be for a sample entry, the source may be some sort of lightsource, the sample compartment may be where the measurement is madeafter reacting with the coating, the optics may be used to transfer andmodify the signal prior to detection, the detector may be a transducerthat converts an optical signal into an electrical signal that could beread by a computer, and the output may be a computer that converts theelectrical signal from detector and performs analysis on the data toproduce a result. The result may be a simple detection, a quantitation,or even a full identification of the amount of analyte in the sample.

FIG. 3 is a schematic showing prior art and the current invention.Bottom) the prior art shows molecules attracted to coatings through weakintermolecular forces. Top) the current invention shows bonding of ananalyte to the coating. Here the analyte may be localized at the coatingthrough an intramolecular force (e.g. a chemical bond) that stronglyholds onto the analyte.

FIG. 4 is a schematic representation of how the detection system maywork on a molecular basis, and includes a depiction of a sensor molecule(and some subunits), its attachment to a surface, and its subsequentreaction with an analyte.

FIG. 5 shows some abbreviated chemical structures illustrating the typesof acyl carbon derivatives which could be used as the RFG portion of asensor molecule.

V. BEST MODE OF CARRYING OUT THE INVENTION

As can be easily understood, the basic concepts of the present inventionmay be embodied in a variety of ways. It involves both analysistechniques as well as devices to accomplish the appropriate analysis. Inthis application, the analysis techniques are disclosed as well asvarious devices described and as steps which are inherent toutilization. In addition, while some devices are disclosed, it should beunderstood that these not only accomplish certain methods but also canbe varied in a number of ways. Importantly, as to all of the foregoing,all of these facets should be understood to be encompassed by thisdisclosure.

The general spectroscopic technique can be easily understood fromreference to FIG. 2. FIG. 2 shows an analyzer (1) which has some type ofinput (2) and results in some type of output (3). Either by providingthe analyte or controlled substance itself or by providing an inputwhere some type of sample surface or sample (4) can be provided to theanalyzer (1), input (2) facilitates sample (4) to be provided withinanalyzer (1). Since the focus is upon analysis through spectroscopy,analyzer (1) as well as the various components are configured forspectroscopic analysis. Either within or external to analyzer (1), sometype of source (5) may provide one or more wavelengths of energy towhich a spectroscopic sample (4) is exposed. The source (5) may be alaser or some other type of highly regulated light source. It may alsobe some type of broad spectrum source. Typically, the source (5) hasbeen highly regulated to provide a constant output. In keeping with oneof the aspects of the present invention, less expensive sources mightnow be used. In general, however, the spectroscopic sample (4) may beprovided so that the source (5) can be exposed to it in a manner that atleast one incident wavelength of energy can impinge upon the sample (4).In this way source (5)—which may be virtually any type of illuminationdevice—can illuminate the sample (4). Since most spectrographic analysiswhich is of interest in the present invention involves the analyte beingon some type of surface, sample (4) may configured as a spectroscopicsample surface. As such the sample has the appropriate characteristicsfor a particular spectroscopic technique. In the situation of SurfaceEnhanced Raman Scattering, the spectroscopic sample surface may havesuch characteristics as those skilled in the chemical arts readilyunderstand so that the spectroscopic sample surface can interact andprovide an appropriate Raman scattering or surface enhanced Ramanscattering as the case may be. The spectroscopic sample surface mayinclude on it some type of event location which may either be the entiresurface or singular locations (such as wells) so that many differentanalytes or sensors can be utilized.

In keeping with the present invention, the spectroscopic sample surfacemay be adapted so as to retain the analyte through some type ofattachment link. While in the prior art, this attachment link may haveinclude primarily weak interactions, in the present invention asignificant difference may be provided so that the attachment linkcreates either a permanent link or perhaps a covalent link in somemanner. Regardless of the specific type of link, the attachment link isdesigned so that at least at one spectroscopic sample location theanalyte or controlled substance can be linked in some manner which canbe spectroscopically sensed, that is, in a manner so that an appropriatespectral change exists as a result of the analyte's presence. Asignificant departure by the present invention with respect to theteachings of the prior art is that this link may retain the analyte—orat least some component chemical or other aspect of the analyte—throughformation of a covalent bond, or stronger interaction.

When the source (5) provides at least one wavelength of energy, theincident wavelength of energy can be affected in some manner at least inpart by the analyte as it is retained on the spectroscopic samplesurface. This broad possibility, namely, the fact that it merely affectsthe spectroscopic signal means that the signal can either be a totallynew signal (such as that of an adduct), or it can simply alter thesignal in some indirect manner by the presence of the analyte. Thealteration then results in a spectroscopic signal which is typicallychanged emission or adsorption wavelength(s) of energy so as to producea signature which can be analyzed and determined uniquely as a result ofthe presence of the particular analyte desired. The sample (4) caneither have on it, or be itself, some type of sensor molecule whichinteracts with the analyte. The changed wavelength or wavelengths maythen be further focused or conditioned by optics (6) so that some typeof detector (7) can be positioned to receive the changed wavelength orperhaps the spectroscopic signal. This signal can then be provided forsome type of analysis.

The analysis may consist of a computer determination through comparisonsoftware or some other determination, all as is well known, to gaininformation with respect to the analyte or controlled substance. Thus,the analyzer (1) acts so as to spectroscopically analyze the signal.This analysis may be as simple as comparisons to known spectra or otherdata which may be stored in a computer or other such device.

As mentioned, it may be desirable to have a spectroscopic samplesurface. As those skilled in the chemical arts readily understand, forRaman spectroscopy this spectroscopic sample surface may actually be aRaman spectroscopic sample surface or a Raman surface, namely, a type ofsurface which provides the appropriate interactions to achieve the Ramanscattering phenom-enon. Similarly, for Surface Enhanced Raman Scatteringspectroscopy this spectroscopic sample surface may actually be a SurfaceEnhanced Raman Scattering spectroscopic sample surface, namely, a typeof surface which provides the appropriate interactions to achieve theSurface Enhanced Raman Scattering phenomenon. Likewise, the illuminationsource may be a specific Raman illumination source, namely, the type ofsource appropriate for Raman Spectroscopy so that Raman illumination isexposed to the sample (4) to produce Raman scattering or a SurfaceEnhanced Raman Scattering illumination source. It should also beunderstood that the surface, while typically a somewhat planar solid,may also include suspended particulates and the like so long as anappropriate Raman or other sensor molecule type is included to permitthe interactions desired.

As mentioned earlier, the focus on spectroscopic analysis differentiatesthe invention from other separation types of techniques. Such techniquesmay include chromatography or other aspects which, although oftenincidentally used, are fundamentally different from the field ofspectroscopic analysis. Chromatographic techniques often simply separatematerials on the basis of their interaction with a surface. Here, earlychromatography used paper, activated charcoal, or alumina to separatepolar materials from a nonpolar solvent phase. This allowed theanalytical chemist to remove polar interference from a nonpolar analyteor to sequester a polar analyte onto a stationary matrix and flush awaynonpolar interferences. As the chromatographic field has advanced, it ispossible to separate materials based on smaller and smaller differencesin affinity for the solid stationary phase. More recently, the field ofliquid chromatography has improved through the use of affinity coatings.The coatings allow one to chemically modify the solid stationary phasesuch that it possess affinities different from the uncoated material. Aclassical example is the coating of silica which is a very polar surfacewith an alkylsilane to produce a very nonpolar surface. This makes itpossible to partition nonpolar materials onto the stationary phase andto have a polar solvent phase (mobile phase). This form ofchromatography is called reverse phase chromatography. The realizationthat coatings could be used to modify the chemical properties of thesolid phase led to the development of charged coatings. These are usedfor yet another form of chromatography called ion chromatography. Thisform of chromatography utilizes Coulombic attraction and polarizationforces to separate charged species from a noncharged mobile phase. Allof the above described chromatographic methods use an interfacial forcesfor separation. While they may be incidentally used in the preparationof samples for spectroscopic methods, they are not themselves aspectroscopic analysis technique. Similarly, affinity chromatographywhich is often used to isolate or purify enzymes, may involve transientbonding to substrates, however, again, this technique is primarily aseparation technique and is not designed for spectroscopic analysis. Thetransient bonding is of necessity nonpermanent as it is simply anintermediate step of enzyme activity and is designed to be released.

Although the prior discussion with regards to spectroscopic techniquesfocused upon the utilization of SERS and Raman technology, it should beunderstood that a variety of detections systems could be employed withthe present invention to determine the concentration or characteristicsof the analyte. At present it simply appears that the most attractive isSurface Enhanced Raman Spectroscopy (SERS) because of the tremendousincrease in Raman signal intensity associated with localizing a moleculenear a silver, copper, or other appropriate metal surface. SERS is alsoattractive due to the high information content associated with Ramanspectroscopy, which assays the transitions between vibrational states ofa molecule. Infrared (IR) spectroscopy, which also detects vibrationaltransitions in molecules, is also a good candidate for a detectionsystem. Because the sensor coating and subsequent adduct coating areboth surface bound, the most probable application of IR spectroscopywould be in the context of direct-, or indirect-reflectance Fouriertransform infrared spectroscopy (FTIR), in which the lower sensitivityof the technique could be compensated by the multiple acquisition andsubsequent analysis of spectra. Other optical techniques which might beapplied to measuring concentrations of the surface bound species arefluorescence (or photorescence) spectroscopy, or the measurement offluorescence (or phosphorescence) lifetimes. Non-optical techniqueswhich could be utilized for these measurements could includeelectrochemical methods (e.g., voltametric or amperometric methods). Inthe simplest cases (e.g., those in which only a single analyte is likelyto be encountered), any other method (spectroscopic or nonspectroscopic)which can be used to characterize the presence of a species at a surfacecould also be utilized, provided that it could distinguish between thesensor coating and the adduct coating. More demanding applications(i.e., more than one possible analyte) would probably require atechnique which could distinguish between the sensor coating and each ofthe possible adduct coatings.

As disclosed, the coating may consist of a variety chemical classes.Carbon containing groups such as those having some carbon atom in theirstructure and even non-protein groups of coatings could be used as thereactive coating. As contemplated in one of the preferred embodiments, adiazonium compound or some derivative of that compound could be used asa sensor coating. The diazonium compound may also include a diazoniumsalt sensor coating. Any diazonium compound sensor coating could beselected in situations in which the particular analytes involved wouldreact appropriately with the diazonium compound sensor coating in orderto provide the desired spectrum. Thus, a controlled substance whichreacted appropriately would be considered a diazonium-specificcontrolled substance. In this manner at least some chemical of thediazonium-specific controlled substance would interact with theanalyte-reactive coating (8). Some chemicals which represent bothcontrolled substances and appear to be appropriate as adiazonium-specific controlled substance include codeine, heroin,psylocibin, tetrahydrocannabinol and morphine.

Some other specific coatings in keeping with this aspect of the presentinvention may include activated acyl compounds and their associatedacyl-specific controlled substances or analytes. The substances mayinclude not only those mentioned earlier, but also some alcohols,amphetamines, or methamphetamines. Nitroso-sensor coatings may also beused with nitroso-specific analytes as is mentioned later.

As discussed in more detail later, any of the coatings may be placed onthe desired surface through techniques easily understood and may thus beused to sense the appropriate substance. This substance may range fromgeneral analytes to the controlled substance application mentionedearlier to medical applications as well. In that latter instance, bloodsubstances, namely, those being blood itself or coming from or derivedfrom blood products may be used for detection of either controlledsubstances or medically important information such as bilirubin or thelike. In an analysis, even finger printed or complex spectralcharacteristics could be provided or used within computer or othercapabilities so that individualized determinations might be made. Otherapplications beyond those mentioned range from mining to environmentalapplications. Naturally many of these substances would beconsideredcontrolled substances—that is, they are regulated in some way, be it dueto illegal drug use, environmental concerns, or for other reasons.

As mentioned earlier, the linking between the analyte and the coating onthe surface of the sample (4), may be selected so as to achieve notmerely permanent bonding of the coating to the surface, but,significantly, permanent bonding of the analyte to the coating. Unlikethe weak interactions considered appropriate in the prior art, thislatter permanent bonding can include links which are merely retained toa significant degree, links which might be truly nonremovable inordinary use, or even some type of intermediate level of linking. Thebonding may include, of course, a covalent bonding such as those skilledin the chemical arts readily understand to be highly permanent or suchas may even create a new molecular species. Such a species, namely, thecombination of the analyte or controlled substance and the coating canbe an adduct or even a covalently bonded adduct. In this embodiment evenif a new species (or more importantly spectral signature) is created,the invention recognizes that the original spectral characteristics ofthe analyte need not be necessarily retained, but rather the spectralcharacteristics of the adduct may be adequate. Thus an adduct of anykind whether permanent or not, may to some degree present a significantdifference over the prior art. Thus, by selecting a coating which isadapted to form a covalent bond with a particular analyte, the originalsignature of the analyte may be lost, however, the new signature—that ofthe covalently bonded adduct might now be present. In creating an adductthe spectroscopic signal present may now be very different and may noteven be an analyte signal but rather an adduct-specific signal which isproduced by the treated surface.

A coating as described above may be specifically created through the useof some sort of reactive functional group which would itself be adaptedto form the desired adduct. As is mentioned later, an interesting aspectis that through this technique even slight variations in the reactivefunctional group of the coating can give very different spectralsignals. For instance, simply relocating the functional group on ascaffolding or creating different types of bonds can be used to alterthe spectral characteristics and yield a very different spectrum fordifferent analytes or different circumstances.

By including the possibility of creating an adduct as opposed to weaklylinking the analyte to the surface, a situation where the equilibriumconstant for locating the analyte or adduct at the surface essentiallyat infinity can be created.

As mentioned earlier, one of the advantages of utilizing permanent orperhaps covalent bonding of the analyte or controlled substance to thespectroscopic sample surface (4) is that once these bonds have beencreated, they may not be easily removed or broken. Thus it may now bepossible for some analytes to wash the surface with a liquid either toremove excess analyte, to stop the continued creation of the bonding ofthe analyte over a period of time, or to remove interfering substances.With respect to stopping the citation of additional bonding, a greaterdegree of accuracy may now be possible in that the sample surface priorto being presented to the analyzer (1) maybe washed at a specific timeto completely stop any continued creation of the covalent bond. Thismight also have the added benefit (or independent benefit) ofsimultaneously removing any interfering substances which can be washedaway. Similarly, it is now possible to utilize this washing technique toremove my excess analyte. Again, this may or may not occursimultaneously or may occur independently from the steps of stopping thecreation of the covalent banding of the analyte to the coating or thestop of removing interfering substances.

In yet another embodiment of the present invention, the coating may bedesigned to present an internal standard. By internal standard it ismeant that the coating would separately present an unaltered signalwhich could be sensed to yield information regarding the real timestatus of the source (5) so that variation in the illumination ordetection functions could be factored into the analysis and compensatedfor without a need for a more expensive laser or other illuminationsource (5). This internal standard could be a lower or even separateportion of the coating itself. It could also be from a tether group. Assuch it could present an internal standard which was co-located with theadduct or co-located with the chemical reaction event location (9) sothat a singular illumination process could achieve the signal of both aspectrum of the adduct as well as a spectrum for the internal standard.Examples of such an internal standard could include a carbon chainstructure, aromatic ring, non-reactive chromophore, a nitro group, asulfonyl, a non-carbon structure, or other such items as those skilledin the chemical arts would readily recognize. Such an internal standardcould be specifically included in the design of the coating itself.

The construction of the analyte reactive coating (8) may be achieved ina variety of ways depending on the specifics of the application. Onetype of design is illustrated diagrammatically in FIG. 4. As shown, asensor molecule possessing a tether (TETH) could be terminated by asurface attachment group (SAG), a reactive functional group (RFG) andpossibly even one or more modifier group(s)(Z). These could be placed ona suitable surface to give the sensor coating. In one of the otherembodiments, a precursor molecule having a tether, perhaps a modifier(s)Z, and a RFG-precursor (RFG) may be used to coat a suitable surface. Byincluding a precursor arrangement, the coating might be designed so thatin a subsequent step, the RFG′ could be transformed to the RFG to yielda fully reactive sensor coating.

In use, the sensor coating could be exposed to the analyte (A). Thisanalyte may be contained within a solid, ligand or gas under conditionswhich might allow a chemical reaction to take place between the sensormolecules and the analyte. After an optional rinse of the new adductcoating to remove possibly interfering substances or interferinganalytes (IA) which do not react, the adduct coating could then beassayed by an appropriate spectroscopic technique to quantify the amountof adduct and/or remaining sensor coating present. Aspects of each ofthese steps are discussed in detail below.

As discussed above, fine sensor molecule may incorporate a tether, areactive functional group (or its precursor), and in some instances, amodifier group. All of these groups may be attached to a centralmolecular scaffolding (CMS).

Since the surface attachment group is designed to hold the coating tothe surface, the choice of SAG would likely depends on the surfaceinvolved, as well as on the functionality present in the sensor molecule(see below for a discussion of the latter issue). In each instance thesurface attachment group, namely, the portion of the molecule whichwould serve to link atoms in the sensor molecule to the surface could bechosen based upon the type of substrate or surface which is desired tobe used. The following discusses a variety of such surface materials;naturally others could be used and in such instances, the SAG wouldlikely be varied as those skilled in the chemical arts would readilyunderstand.

Metal surfaces. Likely metal surfaces include silver, gold, copper andmercury. A particularly appropriate SAG for these surfaces appears to bethe thiol group RSH (or RS—) or some derivative of the thiol group suchas RCOSM, RCOS₂M. ROCOSM, RSCOSM, RSCS₂M, ROP(S)_(m)(O)₃-_(m)M₂,RSP(S)_(m)(O)₃-M₂, RSSR′, RSSSR′, RSNR′R″, in which R=RFG-Ar-spacer-,M=H or metal ion, and R′,R″=alkyl or aryl or H. Other SAG's which mightbe appropriate are the polar groups RSO₃M, RSO₂M, ROSO₃M, ROSO₂M, RCO₂M,ROCO₂M, RNR′CO₂M, ROP(O)O₂M₂, RP(O)O₂M₂.

Silica, alumina, or florasil surfaces. In addition to the polar grouplisted for metal surface above, polar SAG's likely appropriate forattachment to these surfaces include RR′R″N, RR′R″R′″N+, and thecorresponding phosphine and phosphonium salts in which P replaces N.Other SAG's include RSiX₃ (X=leaving group such as halide, inorganicester, and R, R′, R″, M defined as for the metal surfaces above), andRSi(OR′)₃.

Ion exchange resins. Any of the negatively charged SAG's listed underthe metal surfaces section above (is., RCOSM, RCOS₂M, ROCOSM, RSCOSM,RSCS₂M, ROP(S)_(m)(O)₃-_(m)M₂, RSP(S)_(m)(O)₃-mM2, RSO₃M, RSO₂M, RSO₂M,ROSO₃M, ROSO₂M, RCO₂M, ROCO₂M, RNR′CO₂M, ROP(O)O₂M₂, RP(O)O₂M₂) could beloaded outo an anion exchange resin. Amine, ammonium, or phosphoniumsalts could be loaded onto cation exchange resins. While coatings ofthis type would be particularly appropriate for use with analytesolutions not containing potentially competing ions, it may still bepossible to utilize these systems for ion containing solutions, sincemany of the SAG's listed above have particularly high affinity for ionexchange resins, and because appropriate tether design could hinder theexchange rate with undesirable ions.

Plastics. Any of a number of functionalized polymers could be modifiedwith an appropriate SAG. For example, chloromethylation of a polystyrenebased plastic could be followed by reaction with RSM, ROM, or RNR′R″.Alkyl acrylate or alkylmethacrylate polymers could be subjected to esterexchange with ROH, or aminolysis with RNR′H. It may also be possible toadhere a hydrophobic tether group to a hydrophobic plastic throughsimple hydrophobic attraction.

Cellulose. An example of this class of surfaces is filter paper. Thiscould be modified by any type of activated carbonyl compound (e.g. RCOX,RNCO, R and X defined as in the metals and silica sections above).Modification of specialized cellulosic material (e.g., cyclodextrins)offers the possibility of unique selectivities towards certain analyteclasses.

Coating Methods. There a number of possibilities to create the coating.While many of these are easily understood by those of ordinary skill inthe chemical arts, two general approaches may be noteworthy in theformation of a sensor coating: application of RFG-Ar-tether-SAG to givethe sensor coating directly, or application of RFG′-Ar-tether-SAG, inwhich RFG is a group which must be transformed to the desired RFG in asubsequent activation step. The advantages of the first approach are atleast two-fold. The prior activation/formation of the RFG should bestraightforward, since the reactions leading to it should generally bestandard solution phase chemistry. In addition, this approach shouldtypically yield the sensor coating directly. There are, however,potential disadvantages of this approach for some classes of senorcoatings and/or SAG's. These disadvantages include the possibility thatthe sensor coating may not have a long term stability (e.g., due toreaction with adventitious water, oxygen, light), and the possibilitythat a particular combination of RFG and SAG may not be chemicallycompatible, or that the RFG may not be compatible with the surfaceduring the coating process (due to the geometry of attachment of thesensor molecule and tether, it could be possible that one could haveRFG's which could react with a surface in the process of coating, butcould not react once attached to the surface). While it is likely thatthe potential problem of sensor coating stability could be solved simplyby appropriate protective measures (e.g., a dissolvable overcoating, orthe use of otherwise hermetically sealed surfaces), the problem ofSAG/RFG incompatibility may require the second approach, in which theRFG is formed only after surface coating. In addition to avoiding theproblem of functional group incompatibility, this approach may bedesirable from the standpoint of sensor coating stability. In practice,a stable pre-sensor coating could be prepared, stored for someindefinite period of time, and then the RFG′ converted to the RFG byactivation immediately prior to exposure to the analyte. A potentialdrawback to this approach is the possibility that the desired RFGactivation might not occur efficiently on a surface. In some cases, thefeasibility of this approach might even need to be determined on a caseby case basis. Other approaches may be appropriate depending on marketplace and other considerations and should be relatively easily designedonce the basic principles are understood.

Again, it should be understood that each of the above examples representmerely come choices available depending upon the particular surfaces orother aspects involved in a particular application. Variations should beunderstood as encompassed within this disclosure since with the basicteachings those of ordinary skill in the chemical arts would be able toachieve variations in any of the components mentioned without undoexperimentation.

Attached either directly to the surface attachment group or through sametype of tether, may be a central molecular scaffolding. The CentralMolecular Scaffolding may serve to physically hold the variouscomponents of the sensor molecule in place. It may also play a role inthe analysis of the adduct coating. This could be particularly true inthe case of spectroscopic techniques which rely on fluorescence, orwhich require detection at long wavelengths, for which polycyclicaromatic scaffoldings might be particularly well suited. If the modifierZ is to have a direct influence on the reactivity of the RFG, or uponthe spectroscopic characteristics of the adduct coating, it mightpreferably be desirable to have a direct electronic “connection” betweenZ and the scaffolding, and between the scaffolding and the RFG (andsubsequently the analyte portion of the adduct). Once again, this mightsuggest a central scaffolding which might likely be a monocyclic orpolycyclic aromatic (or heteroaromatic) ring. For sensor coatings whichprimarily act by ensuring the propinquity of target analyte to thesurface, it may not be useful to have the electronic connectiondiscussed above. For these system. It may be possible (though notnecessary) to employ non-aromatic scaffoldings. In general, however,examples of some central molecular scaffolding groups may includesimilar types of rings as mentioned earlier with regard to the surfaceattachment group, namely, the establishment of the monocyclic aromaticring, a polycyclic aromatic ring, a heteroaromatic ring, or anon-aromatic scaffolding as well.

As mentioned earlier, one of the advantages of an embodiment of thisinvention is the possibility of removing undesired substances whichmight have spectral characteristics which interfere with analysis of thetarget analyte. This may be accomplished by washing the surface layersubsequent to reaction, thus removing the unreacted, interferingsubstances. From the above description, it may be gathered that thisgeneral approach to analyte specificity and enhanced signal-to-noise isnot expected to be limited to a specific type of surface, though certainof the spectroscopic methods used for final analysis may requirespecialized substrates (e.g., silver, gold or copper for use with SERS).

As previously discussed, the surface attachment group may be linked tothe central molecular scaffolding through some type of tether. Thetethering group attached to the sensor molecule may simply be zero tomany carbon or non-carbon atoms in a chain (or incorporating a ring)terminated by a SAG which is appropriate for attachment to the chosensurface. Alternatively, the tether may incorporate a nonreactingchromophore of some sort (e.g., some type of aromatic ring). Suchincorporation could allow for the possibility of using the tetherchromophore as an internal standard for quantification of thespectroscopic changes on going from the sensor coating to the sensoradduct coating. Tethering chains incorporating aromatic or long-chainaliphatic carbon chains (or perfluorinated carbon chains) also may proveto be advantageous from the standpoint of stability of the sensorcoating, since the hydrophobicity of these groups could tend to protectthe surface attachment points from the solvent and dissolved solutes. Inthe ease of detection systems designed for use with nonpolar solvents(e.g., one of the activated acyl based sensor molecules), a polar,nonreacting tether could be utilized. Examples of this type of tetherwould likely be amide based (e.g., a polypeptide, possibly terminatedwith cysteine), though other polar tethers could also be employed (e.g.,based on sulfonates or sulfoxides). A tether design which would beespecially effective in masking a surface from an organic solvent wouldbe one of the type RFG—Ar—(CH₂)_(n)—N+R₂(CH₂)_(m)—SAG Y—(Y—=counterion),since the ionic interface could effectively preclude migration ofnonpolar molecules.

Functionally, an important aspect of the coating may be the inclusion ofsame type of reactive functional group, namely, a group of atoms whichacts in such a fashion as to react with the analyte and link the analyteto the coating. The coating itself may thus be adapted to be attached tothe analyte of desire. This attachment may occur through a covalentbond, the mere creation of an adduct, some type of permanent link, orotherwise. Because of the importance of this structural feature of thesensor molecule, and because of the differences in analyte specificitywhich result from difference in the nature of the RFG, its location andthe like, the various classes of RFG's are discussed with somespecificity below. General examples of reactive functional groups whichcan be used include not only the diazonium groups, azo species, andnitrous, but also activated acyl sensors as well as non-proteinmolecules.

Diazonium. The diazonium group is probably the most interesting andpromising of the RFO's due to its high reactivity towards certainclasses of molecules, as well as the nature of the adduct it can formwith these molecules. While the high reactivity of diazonium salts iswell known, and is responsible for their utility in the preparation ofdiverse functional derivatives of aromatic rings, these ions are notindiscriminately reactive. Thus, proper choice of reaction conditionsfor their formation leads to diazonium salts which can be kept for longperiods in solutions and subsequently reacted with an added reagent, oreven isolated and stored for long periods of time prior to redissolutionand reaction. One of the characteristic reactions of diazonium salts isthe addition of a nucleophile (Nuc) to the terminal nitrogen of theArN₂+ group to give azo species Ar—N═N—Nuc. As a consequence of the highdegree of conjugation and polarizability of these azo species, they arein many cases dyes, and appear especially well suited by virtue ofwavelength of maximum absorption (λmax) and/or extinction coefficient(ε) to detection by any of a number of techniques. Alternative pathwaysfor nucleophiles involve substitution (directly, or indirectly throughthe intermediacy of an azo species) of the N2+group to give a newaromatic species. Though the new species often may not have as desirablespectral characteristics as the azo type products, the replacement ofthe unique N2+ chromophore by a different group typically is distinct.One of the classical reactions of diazonium salts, that can result inthe formation of aryl chlorides and aryl bromides, follows a radicalpathway demanding the presence of copper (or other metal) salts, and maybe unlikely to occur except under specially designed conditions.

Nitroso. Aryl nitroso compounds are expected to share many of the samedesirable features of diazonium salts in terms of their reactivity withselected classes of potential analytes, and in terms of the desirablespectral characteristics of the resulting adducts. Aryl nitrosocompounds may react with amines to form azo compounds (Z—Ar—NO+RNH2gives Z—Ar—N═N—R) which, as indicated above, sometimes form the basis ofmany dyestuffs. If an electron rich phenol or aromatic amine isemployed, then nitroso compounds may react to form quionimine, orquinodiimine type structures. These reactions can be exemplified by theformation of the dye gallocyanine (from gallic acid andp-nitrosodimethylaniline) and Nile Blue A from 1-naphthylamine.

Activated Acyl Sensors. This class of RFG or sensor molecules comprisecompounds having a reactive acyl type carbon. In addition to compoundsin which the reactive carbon is in the oxidation state of a carboxylicacid (e.g., acid chlorides, imino esters, iminium esters, etc.), thisclass of compounds should be understood to include compounds in whichthe reactive carbon is in the oxidation state of carbon dioxide (e.g.,carbonyl halides (ROCCO), isocyanates, diimides, etc.). Examples areillustrated in FIG. 5, along with the structures of the adducts expectedupon reaction with alcohols and amines (RXH, X═O and NH or NR′,respectively). All of these compounds are expected to be highly reactivetowards water, and thus it is likely that sensors based on this type ofreactive functional group will be of greatest utility for directlyassaying non-aqueous and non-alcoholic solutions. Exceptions to thisgeneralization may, of course, occur if the analyte of interest is ofsufficiently great reactivity (and sufficiently high concentration) thatit may compete with water/alcohol for the RFG. An alternative todirectly assaying an aqueous sample might involve extraction of theanalytes of interest from an aqueous solution into an organic solvent,followed by exposure of the extract to the sensor coating. Similarconsiderations as those discussed above for the preparation of thesensor coating may apply here as well. Although direct formation of asurface from a molecule incorporating the desired RFG may have somepractical benefits, depending on the specific identities of the RFG andSAG, it may be necessary or advantageous to prepare a coating havingfrom a molecule which has a group RFG′ that may be unreactive, but whichmay be transformed to the RFG after formation of the coated surface.

Both the diazonium and activated acyl sensor coatings might be used forthe detection of controlled substances and the like. Particularly theactivated acyls may also be used for alcohols and amines as well. Thenitroso coatings though likely not as applicable for drug sensing alsomay offer specific chemicals for which they are appropriate. In general,the sensor coatings may include a wide variety of general type andclasses of molecules such as those including a halide, anelectron-donating group, an alkoxy group, amide group, amino group, anelectron-withdrawing group, a nitro group, a carboxlic acid group, anester group, a sulfoxide group, or a sulfone group, diazonium salts,electrophilic carbons, or a carbonyl group.

The large variations in reactivity of aromatic compounds with aryldiazonium salts may allow a significant degree of selectivity to beachieved in the analysis of mixtures of compounds. In a gross sense,aromatic analytes may be divided into electron rich and electron poorgroups. Aromatic compounds more electron poor than benzine are notexpected to react with Z—Ar—N2+, though exceptions may be possible.Electron rich analytes susceptible to analysis through diazonium basedsurface coatings include phenols, diaryl ethers, aryl alkyl ethers, arylesters, aryl amines (anilines), aryl amides (ArNRCOR′), pyrroles,furans, thiophenes, and any other electron rich heteroaromatic ringsystem. For all of these compounds, the direct conjugation of the twoaromatic rings through the diazo group may mean that adduct s could havevery unique and distinguishable spectral signatures.

To further permit variation in the reactive functional groups end theanalytes for controlled substances desired to be sensed, it is possiblethat the reactive functional group may be first presented as a reactivefunctional group precursor. In this fashion the group itself would notact at its initial configuration to achieve the reaction with theanalyte but rather would require same type of activation step to convertit to its reactive form. Such may be presumed by subjecting the reactivefunctional group precursor to an activation substance. In this mannerthe reactive functional group precursor might be activated to produce areactive functional group, namely, a substance which in its activatedstate would act to react with a desired analyte. Thus, the reactivefunctional group precursor might be activated to become the chemicalreaction event location by being subjected to some type of activationsubstance. Examples of reactive functional group precursors include: butare not limited to aryl amines (for diazonium, RFGs), aryl amines orhydroxylamines or nitro groups (for nitroso RFGs), and carboxylic acidsor alcohols or amines (for activated acyl groups RFGs). Examples ofactivation substances include: sodium nitrite, thionyl chloride, and thelike as those skilled in the chemical arts would readily understand.

To permit even further variation, for certain classes of substrates, itmay be possible to modify reactivity (and thus, selectivity in a samplecontaining mixed analyte classes) in either a positive or negative sensethrough manipulation of the reaction conditions. This strategy may beillustrated by considering a mixture of a phenol (ArOH), aryl ether(ArOR, R either aryl or alkyl) and an amine (ArNR2). At pH 7 all ofthese compounds are expected to be in neutral forms given above, and thebetter donor ability of nitrogen as compared to oxygen should result ina reactivity order of ArNR2>ArOH≈ArOR, allowing for selective/enhancesdetection of the aryl amine. At higher pH (>8-9), significant ionizationof the phenol may generally take place to give phenolate, ArO—.Phenolate ions are among the most reactive species known towardsdiazonium salts, often showing near diffusion controlled rates ofreaction. Thus, at conditions of high pH, the reactivity order isexpected to be “ArOH”>ArNR2>ArOr. Indeed, selectivity of this type hasbeen seen in the case, of the reactions of 5-dimethylamino-1-naphtholwith benzenediazonium ion. In this case, reaction occurs para to thedimethylamino group at low pH, and ortho to the oxygen at high pH.Finally, if exposure of a sensor coating were to take place at very lowpH (e.g., pH≦3), the amino group of the arylamine would be in aprotonated state (ArNR2H+), rendering the aromatic ring highly electrondeficient and unreactive. Under these conditions the reactivity ordershould be ArOH═AOR>“ArNR2”, allowing the assay of phenols and arylethers in the presence of the normally more reactive aryl amino.

It is possible that attachment of fine RFG and tether/SAG units to acentral molecular scaffolding may result in a coating which reacts at adesirable rate with target analysis to give adducts having desirablecharacteristics from the standpoint of subsequent analysis. If theseconditions am not met, however, it may be desirable to incorporate amodifier group, Z. The modifier (if employed) could be chosen so as toinfluence the reactivity of the RFG, although it may also play a role inenhancing the spectral characteristics of the adduct coating (e.g., interms of λmax, extinction coefficient). In order to increase reactivity,it is likely that an electron withdrawing group would be chosen (e.g.,nitro, carboxylic acid, ester, sulfoxide, sulfone). The nitro groupillustrates the dual role which a modifier could play. In addition toincreasing the reactivity of either a diazonium or activated acyl basedsensor towards all analyte classes, the nitro group may appearparticularly Raman active, suggesting its utility in a SERS buseddetection system.

Thus, this arrangement could present a first modifier and a secondmodifier which could be attached to some aspect of the coating toachieve the desired result. In one regard the modifier might serve toreserve a position on the molecule to react with the controlledsubstance or the analyte involved. In another regard the modifier mightserve to influence the reactivity between the reactive functional groupand the analyte. This influence might actually serve to tune orinfluence the reaction to a desired degree. In one regard such tuningmight serve to provide different ranges or concentrations at which theanalyte reactive coating (8) would react. Thus, the sample (4) might beconfigured with a variety of sensor locations for each to serve as asensor appropriate to a different range of concentrations. It should benoted in this regard that in other aspects of the invention multipleseniors can be essentially co-located at one sensor site to potentiallyachieve a similar type of capability. Regardless, the modifier mightserve to create a group which could be adapted to influence the activitybetween the coating and the analyte to some desired degree.Interestingly, the modifier through its location on the reactivefunctional group or some other aspect can set different ranges based onthe type of modifier chosen. Since these range variations can be manyorders of magnitude different, a great degree of variation can beachieved through this embodiment of the present invention.

Examples of modifiers which may be utilized include electron donatinggroup (oxygen or amine nitrogen or amide nitrogen), electron withdrawinggroups (nitro, carboxylic acid, esters, sulfoxide and sulfones). Asbefore, and as should be understood throughout this application, theexamples should not be viewed as establishing a limitation to theinvention. Under the basic teachings of the present invention those withordinary skill in the chemical arts can easily vary the chemicals toachieve the desired result based upon the type of situation or type ofanalyte involved. Furthermore, a great degree of variations in thevarious substances involved is explained in FIG. 5. This teaching servesas a blueprint for the design of a great variety of chemicals accordingto the present invention.

As mentioned earlier, this invention has applicability in a variety ofinstances ranging from law enforcement to medical to environmentalconcerns. With the teachings of the present invention one need onlyunderstand the particular analyte involved and then utilize theteachings to attempt to design an appropriate reactive functional groupor analyte-reactive coating (8) to achieve detection or analysts.

Without creating a limitation, some of the various analytes naturallyinclude many controlled substances such as codeine, heroin, possiblycocaine, tetrahydrocannibinol (THC), morphine, amphetamines,methamphetamines, or the like. They also include a variety of othersubstances as discussed below. Analytes which should be well suited todetection by diazonium based sensor molecules include the followingclasses and types of substances.

Cyanide. Cyanide can react in a reversible fashion with Z—ArN₂+ to giveZ—Ar—N═N—CN. It as of note that while this reaction is reversible, itinvolves the formation of an adduct which tumults from covalent bandformation. This complex may be orange or red, depending on the geometryabout the N═N double bond. There are two aspects of this reaction whichmay be of practical importance: its reversible nature, and thesensitivity of the reaction to the nature of Z. The reversible nature ofthis reaction means that it may be possible to construct a detectionsystem which may be used multiple times (with appropriate intermediaryrinses) and/or as a flow sensor to detect changing concentrations ofcyanide in a waste stream. The sensitivity of the reaction to the natureof Z offers the possibility of making a detection system which iscapable of quantifying cyanide concentration over a wide range ofvalues. The Hammett equation for this reaction is log(K_(CN))=3.53σ+182, Thus, the equilibrium constant K_(CN) for thereaction ranges from 60,000 M−1 for Z=p−NO₂ to 15 M⁻¹ for Z=p−CH₃. Byemploying mixtures of sensor molecule (each of which may have a uniquespectrum), it may be possible to monitor a wide range at cyanideconcentrations, since levels which may saturate (i.e., overload) asensor molecule having Z=p−NO₂ typically may just be starting toeffectively form adducts with a sensor molecule having Z=m−Cl (for whichK_(CN) is 1,600 M⁻¹). Since the spectra of differentially substitutedadducts may be different, it generally will be possible to distinguishbetween them and determine their concentrations. It should be noted thatsome cyanide bonds—while covalent—may not be totally permanent, suchadducts may not be appropriate for a washing procedure discussedearlier.

Sulphite. The sulfite ion undergoes very rapid reaction with aryldiazonium salts to give the syn isomer of the adduct Z—Ar—N═N—SO₃—. Thisintermediate may then undergo an isomerisation reaction to give thestable anti adduct, or under conditions of high sulfite concentrationmay add a second sulfite to give the bis-sulfite adductZ—Ar—N(SO₃—)NHSO₃—. The rate of the syn-anti isomerization reaction issubstantially slower than that of the initial adduct formation, withhalf-lives of about 250 sec and <2 sec, respectively. Furthermore, therate of this isomerization is not greatly dependent on the identity ofZ. With this relatively slow isomerization, it may be possible todirectly measure sulfite concentrations by spectral assay immediatelyfollowing exposure of the sensor coating to the analyte sample. However,by simply waiting for roughly 30 minutes (which is seven half-lives forthe isomerization reaction), the isomerization reaction may be >99%complete, allowing concentrations to be measured from the spectrum ofthe anti mono-adduct. A further advantage of this approach may be thatthe secondary reaction of the syn- or anti-mono-adducts to give thebis-sulfite may provide a method by which higher concentrations ofsulfite can be measured. Thus, molecules of this type may inherently bedual-range sensors.

Thiols. It may be possible to detect thiols and related species (e.g.,xanthates) through the formation of adducts of the type Z—Ar—N═N—SR. Thepossible competing process of the binding of the thiol to the surface(for mental substrates) could be minimized by having a high level ofsurface coating density for the original sensor coating. In any case,should the thiol bind to the surface, it might be possible to detect itspresence directly by spectroscopic means.

Sulfinic acids. Sulfinic acids react with Z—Ar—N₂+ to giveZ—Ar—N═N—SO₂R, and thus should be amenable to detection by a diazoniumbased sensor coating. The extent of adduct formation, as well as thereversibility of adduct formation appears similar to that found withcyanide. Hence, such potentials for sensor coatings may be similar tothose discussed for cyanide (e.g., reusable, applicable to flow systems,wide dynamic range through use of mixed coatings, etc.).

Amines. Ammonia and primary amines react with Z—Ar—N₂+ to givetriazenes, Z—Az—N═N—NHR (R═H, alkyl, aryl amines are ultimately expectedto lead to C—N coupled products, as discussed below for electron richaromatic compounds). Alternatively, a use of a sensor coating having anoxidizing counterion (such as Br₃—) may lead to formation of aryl asidesZ—Ar—N₃, with ammonia as the analyte. This distinction may allow primaryamines to be analyzed in the presence ammonia by either directobservation and quantification of the final mixed adduct coating(Z—Ar—N═N—NHR and Z—Ar—N₃), or by sequential measurements with twodifferent coatings (one with a non-oxidizing counterion, one with).Other derivative of amines which react with Z—Ar—N₂+ include hydrazinesand hydroxylamine. Both of these types of compounds lead to azides andaryl amines. The fact that the reaction of hydroxylamine can beselectively directed by changes in reaction pH to give azides (low pH)or aryl amines (high pH) offers the possibility of distinguishingbetween the two classes of compounds.

Azide. The azide ion reacts extremely rapidly with Z—Ar—N₂+ to giveZ—Ar—N₃. It thus could be spectroscopically sensed as mentioned above.

Carbon Acids. Compounds of the type RCH₂W, an which W is a strongelectron withdrawing group (e.g., carbonyl, nitro, etc.) react withZ—Ar—N₂+ to form highly conjugated adducts of the type Z—Ar—NH—N═CRW.These reactions are known to proceed through the conjugate base of theanalyte (i.e., RWCH—). Although this might be expected to mean that thisdetection method would be limited to strong carbon acids of the typeWCH₂W′, it appears that while the lower acidity of monoactivated carbonacids RCH₃W (R═H, alkyl, aryl) does indeed lead to loweredconcentrations of the reactive conjugate base, there is a compensatoryincrease in reactivity of the base. Thus, even the relatively unacidicacetone has born observed to couple with benzenediazonium ions undernearly neutral conditions. For carbon acids having only a single acidichydrogen, adducts of the form Z—Ar—N═N—CRR′W at expected to form. Whilethe adducts lack a direct conjugative interaction with the sensormolecule, the —CRR′W group may still be attached in the vicinity of thesurface, and may be susceptible to assay by certain spectroscopictechniques (e.g., SERS, reflectance FTIR) even if it lacks anappropriate chromophore for other techniques (e.g., fluorescence).

Electron Rich Aromatic Compounds. This class of compounds may prove tobe the best suited class of analytes of all those discussed. Thecoupling of aromatic compounds Ar′—D (D=electron donating group) to aryldiazonium salts Z—Ar—N₂+ to give diazo compounds Z—Ar—N═N—Ar′D has beenwidely studied in the context of the production of dyes. The diazoadducts produced in these reactions absorb UV-Visible radiation strongly(i.e., have high extinction coefficients, ε) and typically have theirmost intensely absorbing peak at high wavelengths (i.e., they have aλmax at high wavelengths), making them particularly well suited fordetection by any of a number of spectroscopic techniques. Thesespectroscopic properties lend themselves particularly well tomanipulation through judicious placement of modifier group(s) Z onZ—Ar—N₂+, and by variation of the molecular scaffolding Ar.

Other analytes in general may include phenolic compounds, furans,pyrroles, aryl amines, alcohols, and the like.

Yet another embodiment of the invention involves its teachings to createmultiple sensors. In this regard coatings may be designed which presentat least a first sensor molecule type and a second sensor molecule type.These molecule types would be differentiated in that each type wouldhave its own reaction characteristics or its own differentiatablespectroscopic signal either with respect to the same analyte or withrespect to different analytes. As such the first sensor molecule typecould respond to a first type of analyte in one way to yield a firstsignal, and the second sensor molecule type could respond to a secondtype of analyte in another way to yield a second signal, etc. Naturallya plurality of molecule types could be utilized. In any such design thevarious sensor molecule types might be adapted to bond with differenttypes of analytes, or, more generally, to somehow differently interact.They may even be adapted to interact differently with the same analyteand may, of course, each create their own adducts for spectroscopicanalysis. These adducts would likely yield differentiatablespectroscopic signals in order to permit spectroscopic analysis to gaininformation about either one or multiple analytes.

In multiple sensor arrangements in which different analytes might besensed, either the resulting adduct signal or the coating itself wouldlikely be designed to yield a signal which would provide differentiatinginformation between at least a first a second species of analyte. Inthis fashion, a single sensor could be utilized for multiple analytessimultaneously. It could also be used to provide conditional testing inwhich the presence of one in conjunction with another might yield sometype of conclusion. Similarly, as mentioned earlier with regard to aminetype analytes, sequential testing is also possible. Examples of thistype of arrangement might include physically creating different sensorssuch as having one event location with a non-oxidizing counterion andanother event location with an oxidizing counterion and the like, ormight include separate sensor reaction protocols. Thus in oneembodiment, the step of subjecting the analyte reactive coating (8) toan analyte may perhaps be specifically applied to a controlled substanceor the like to yield multiple adduct types by creating each senormolecule type to achieve a different adduct result. The differentiatedsignals might not only yield information with respect to the merepresence of an analyte, but it also might serve for quantitating thepresence of either or all of the various analytes sensed.

In a configuration where the same analyte might be sensed, it might beappropriate to configure the first and second sensor molecule types sothat their interaction with the same analytes occurred in differentconcentration ranges or with some other different characteristics. Inapplications where different ranges of concentration were chosen, anenlarged range of sensitivity for the sample might be presented for agiven analyte. These different ranges might be separate, such as ininstances where discrete occurrences occurred or might more typically beestablished with awareness of the various saturation levels involved forthe two different types of sensor molecules. In such as applicationwhere the first sensor molecule type had a first saturation level (thatis, the level at which the changes in the signal can no longer beaccurately discerned for variations in the analyte concentration or thelike) and the second had a second saturation level which was higher thanthat of the first, it might be possible to use the first molecule upuntil it was near its saturation level and then to use the second sensormolecule type at a continued, expanded range. Furthermore, in instancesin which an overlap region for the two sensor types might be present, itmight be possible to provide verification of readings in this area viaseparate interaction mechanisms so that one could combine both anenlarged range and a verification of readings. In a more general sense,it would thus be possible to establish complementary ranges, namely,ranges (whether overlapping or not) which complimented each other for agiven application to achieve a desired result (whether verification,expanded range, or otherwise). The first and second analytical rangesmight not even be adjacent if the particular application called for suchan arrangement. Furthermore, even though two different types of sensorsmight be provided on a specific coating it is now possible for these twosensors to provide capabilities, whether used or not, so that inpractice one could sense for the presence of different controlledsubstances or the like with just one sensor plate.

In summary, some of the embodiments of the invasion include a surfacecoating comprising highly reactive sensor molecules which chemicallyreact with a given analyte with the formation of a covalent and/orπ-bond to give an adduct with spectroscopic characteristics which areunique to the new adduct. This surface coating may provide the basis ofa detection system which combines high sensitivity with high specificityfor the identification of certain analytes. The surface attachment ofthe sensor molecules allows reaction of the target analyte(s) to befollowed by a rinse, which may remove nonreactive compounds which maypossess interfering spectroscopic characteristics. This methodologycontrasts radically from solution based analyte modification methods, inwhich non-target impurities may substantially decrease effectivedetector signal-to-noise ratios. The formation of a covalent bond (sigmaor π) between the sensor molecule and the analyte to give a new, uniquemolecule (termed the adduct) greatly distinguishes this method fromthose in which hydrogen bonding, or other weak, or perhaps noncovalentinteractions may serve to modify the spectral characteristics of asensor molecule, or to bind the analyte in a location in which itsspectral characteristics may be assayed. In one embodiment, the sensormolecules which comprise the coating may have three components attachedto a central molecular scaffold: a “tether” terminated by a surfaceattachment group “SAG,” a reactive functional group “RFG” which ishighly reactive towards certain classes of molecules, and possibly oneor more modifiers “Z” which may serve to increase or decrease thereactivity of the RFG towards target analytes, or to modify the spectralcharacteristics of the adduct in terms of either wavelength of maximumresponse to a given spectroscopic assay, or in terms of intensity ofresponse to that assay. Variations in the nature of the tether may allowattachment to a variety of surfaces possessing desirable physical orspectroscopic characteristics. Variation of the RFG, and possibly theconditions under which the coating is exposed to the analyte containingsolution, may allow detection selectivity for certain classes ofanalytes. Variations in the modifier group Z and the central molecularscaffold may significantly influence the nature and efficacy of thespectroscopic technique which is used to assay the premium of theadduct. Variations in these groups may also influence the reactivity ofthe sensor molecule.

The discussion included is this application is intended to serve as abasic description from which a great variety of systems can befashioned. The reader should be aware that the specific discussion doesnot explicitly describe all embodiments possible; many alternatives areavailable through application of the teachings of this invention. Italso may not fully explain the generic nature of the invention and maynot explicitly show how each feature or element can actually berepresentative of a broader function or of a great variety ofalternative or equivalent elements. Again, these are implicitly includedin this patent disclosure. Neither the description, the examples, northe terminology should be taken as an intention to limit the scope ofthis patent. Since a variety of changes may be made without departingfrom the essence of the invention, it should be understood that suchchanges are implicitly included within the scope of this invention.

In addition, each of the various elements of the invention and claimsmay also be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of aembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, the disclosure of a “detectionsystem” or “detector” should be understood to encompass disclosure ofthe act of “detecting”—whether explicitly discussed or not—and,conversely, were there only disclosure of the act of “detecting”, such adisclosure should be understood to encompass disclosure of a “detector”or “detection system” (as the context may be appropriate). Similarly,surface “attachment” could include an “attachment element” or“attaching” and vice versa. Such changes and alternative terms are to beunderstood to be explicitly included in the description.

While the invention explains how to apply knowledge from the skills ofpersons in different disciplines, to facilitate this and to facilitatethe creation of the many variations possible, a list of references isprovided in an information disclosure filed with the application. Allthese are hereby incorporated by reference should they be needed. In anyof these references, it should be understood that to the extentstatements in any of the references might be considered inconsistentwith the patenting of this invention such statements are expressly notto be considered as made by the applicant(s).

We claim:
 1. A method for detection of a controlled substance,comprising the steps of: a) establishing a Raman spectroscopic samplesurface; b) placing a diazonium compound sensor coating on said Ramanspectroscopic sample surface; c) subjecting said diazonium compoundsensor coating to a sample; d) retaining a diazonium-reactive controlledsubstance in said sample on said diazonium compound sensor coating onsaid Raman spectroscopic sample surface; e) exposing saiddiazonium-reactive controlled substance retained by said diazoniumcompound sensor coating on said Raman spectroscopic sample surface toradiation having at least one wavelength of energy; f) affecting achange in frequency of said at least one wavelength of energy at leastin part by said diazonium-reactive controlled substance retained by saiddiazonium compound sensor coating on said Raman spectroscopic samplesurface; g) detecting a Raman spectroscopic signal including said changein frequency of said at least one wavelength of energy; and h) analyzingsaid Raman spectroscopic signal to gain information about saiddiazonium-reactive controlled substance.
 2. A method for detection of acontrolled substance as described in claim 1 further comprising the stepof affecting a change in amplitude of said at least one wavelength ofenergy at least in part by said diazonium-reactive controlled substance.3. A method for detection of a controlled substance as described inclaim 2 further comprising the step of detecting a Raman spectroscopicsignal including said change in amplitude.
 4. A method for detection ofa controlled substance as described in claim 1, wherein said controlledsubstance is selected from the group consisting of codeine, heroin,psilocybin, tetrahydrocannabinol and morphine.
 5. A method for detectionof a controlled substance, comprising the steps of: a) establishing aRaman spectroscopic sample surface; b) placing an acyl compound sensorcoating on said Raman spectroscopic sample surface; c) subjecting saidacyl compound sensor coating to a sample; d) retaining a acyl-reactivecontrolled substance in said sample on said acyl compound sensor coatingon said Raman spectroscopic sample surface; e) exposing saidacyl-reactive controlled substance retained by said acyl compound sensorcoating on said Raman spectroscopic sample surface to radiation havingat least one wavelength of energy; f) affecting a change in frequency ofsaid radiation having at least one wavelength of energy at least in partby said acyl-reactive controlled substance retained by said acylcompound sensor coating on said Raman spectroscopic sample surface; g)detecting a Raman spectroscopic signal including said change infrequency of said at least one wavelength of energy; and h) analyzingsaid Raman spectroscopic signal to gain information about saidacyl-reactive controlled substance.
 6. A method for detection of acontrolled substance as described in claim 5 further comprising the stepof affecting a change in amplitude of said at least one wavelength ofenergy at least in part by said acyl-reactive controlled substance.
 7. Amethod for detection of a controlled substance as described in claim 6further comprising the step of detecting a Raman spectroscopic signalincluding said change in amplitude.
 8. A method for detection of acontrolled substance as described in claim 5, wherein said acyl-reactivecontrolled substance is selected from the group consisting of codeine,heroin, tetrahydrocannabinol, morphine, psilocybin, amines, alcohols,amphetamines, and methamphetamines.
 9. A method for detection of acontrolled substance as described in claim 8, wherein said Ramanspectroscopic sample surface comprises a Surface Enhanced RamanScattering surface.
 10. A method for detection of a controlledsubstance, comprising the steps of: a) establishing a Ramanspectroscopic sample surface; b) placing a diazonium salt compoundsensor coating on said Raman spectroscopic sample surface; c) subjectingsaid Raman spectroscopic sample surface to at least somediazonium-reactive controlled substance; d) retaining adiazonium-reactive controlled substance on said diazonium salt compoundsensor coating on said Raman spectroscopic sample surface; e) exposingsaid diazonium-reactive controlled substance retained by said diazoniumsalt compound sensor coating on said Raman spectroscopic sample surfaceto radiation having at least one wavelength of energy; f) affecting achange in frequency of said radiation having at least one wavelength ofenergy at least in part by said diazonium-reactive controlled substanceretained by said diazonium salt compound sensor coating on said Ramanspectroscopic sample surface; g) detecting a Raman spectroscopic signalincluding said change in frequency of said at least one wavelength ofenergy; and h) analyzing said Raman spectroscopic signal to gaininformation about said diazonium-reactive controlled substance.
 11. Amethod for detection of a controlled substance as described in claim 10further comprising the step of affecting a change in amplitude of saidat least one wavelength of energy at least in part by saiddiazonium-reactive controlled substance.
 12. A method for detection of acontrolled substance as described in claim 11 further comprising thestep of detecting a Raman spectroscopic signal including said change inamplitude.
 13. A method for detection of a controlled substance,comprising the steps of: a) establishing a Raman spectroscopic samplesurface; b) placing a nitroso compound sensor coating on said Ramanspectroscopic sample surface; c) subjecting said nitroso compound sensorcoating to a sample; d) retaining a nitroso-reactive controlledsubstance in said sample on said nitroso compound sensor coating on saidRaman spectroscopic sample surface; e) exposing said nitroso-reactivecontrolled substance retained by said nitroso compound sensor coating onsaid Raman spectroscopic sample surface to radiation having at least onewavelength of energy; f) affecting a change in frequency of saidwavelength of energy at least in part by said nitroso-reactivecontrolled substance retained by said nitroso compound sensor coating onsaid Raman spectroscopic sample surface; g) detecting a Ramanspectroscopic signal including said change in frequency of said at leastone wavelength of energy; and h) analyzing said Raman spectroscopicsignal to gain information about said nitroso-reactive controlledsubstance.
 14. A method for detection of a controlled substance asdescribed in claim 13 further comprising the step of affecting a changein amplitude of said at least one wavelength of energy at least in partby said nitroso-relative controlled substance.
 15. A method fordetection of a controlled substance as described in claim 14 furthercomprising the step of detecting a Raman spectroscopic signal includingsaid change in amplitude.
 16. A method for detection of a controlledsubstance as described in claim 1, 5, 10, or 13, wherein said step ofaffecting a change in frequency of said at least one wavelength ofenergy comprises Raman scattering.
 17. A method for detection of acontrolled substance as described in claim 16, wherein said Ramanscattering comprises Surface Enhanced Raman Scattering.
 18. A method fordetection of a controlled substance as described in claim 17, whereinsaid step of retaining a reactive controlled substance to a compoundsensor coating comprises creating covalent bonds with said controlledsubstance over a period of time.
 19. A method for detection of acontrolled substance as described in claims 1, 5, 10 or 13 furthercomprising the step of washing said compound sensor coating on saidRaman spectroscopic sample surface with a liquid.
 20. A method fordetection of a controlled substance as described in claim 19 whereinsaid step of washing said compound sensor coating on said spectroscopicsample surface with a liquid removes at least one interfering substancefrom said compound sensor coating on said spectroscopic sample surface.21. A method for detection of a controlled substance as described inclaim 19 wherein said step of washing said compound sensor coating onsaid spectroscopic sample surface with a liquid removes excesscontrolled substance from said compound sensor coating on said Ramanspectroscopic sample surface.
 22. A method for detection of a controlledsubstance as described in claims 1, 5, 10 or 13 further comprising thestep of establishing a second compound sensor coating on said Ramanspectroscopic sample surface.
 23. A system for detection of a controlledsubstance, comprising: a) a Raman spectroscope; b) a Raman spectroscopicsample surface; and c) a diazonium compound sensor coating on said Ramanspectroscopic sample surface, wherein said diazonium compound sensorcoating retains diazonium-reactive substances in spectroscopic samples,and wherein said Raman spectroscope differentiates saiddiazonium-reactive controlled substances.
 24. A system for detection ofa controlled substance as described in claim 23 wherein said diazoniumcompound sensor coating on said Raman spectroscopic sample surfacegenerates a covalent bond with said diazonium-reactive controlledsubstances.
 25. A system for detection of a controlled substance asdescribed in claim 23 and further comprising an adduct formed by saiddiazonium compound sensor coating on said spectroscopic sample surfaceand said chemical in said controlled substance.
 26. A system fordetection of a controlled substances, comprising: a) a Ramanspectroscope; b) a Raman spectroscopic sample surface; and c) an acylcompound sensor coating on said Raman spectroscopic sample surface,wherein said acyl compound sensor coating retains acyl-reactivesubstances in spectroscopic samples, and wherein said Raman spectroscopedifferentiates said acyl-reactive controlled substances.
 27. A systemfor detection of a controlled substance as described in claim 26 whereinsaid acyl compound sensor coating on said Raman spectroscopic samplesurface generates a covalent bond with said controlled substances.
 28. Asystem for detection of a controlled substance as described in claim 26wherein said Raman surface comprises a Surface Enhanced Raman Scatteringsurface.
 29. A system for detection of a controlled substance asdescribed in claim 26 and further comprising a acyl activator toinfluence reactivity between said acyl compound sensor coating and saidcontrolled substance.
 30. A system for spectroscopic analysis of ananalyte, comprising: a) a Raman spectroscopic sample surface, whereinsaid Raman spectroscopic surface comprises a Surface Enhanced RamanScattering surface; b) at least one Raman spectroscopic sample locationon said Raman spectroscopic surface; and c) a diazonium compound sensorcoating at said at least one spectroscopic sample location; wherein saidanalyte comprises a diazonium-reactive analyte in a Raman spectroscopicsample, and wherein said diazonium compound sensor coating retains saiddiazonium-reactive analyte.
 31. A system for spectroscopic analysis ofan analyte as described in claim 30 wherein said diazonium compoundsensor coating on said Raman spectroscopic sample surface establishes acovalent bond with said diazonium-reactive analyte.
 32. A system forspectroscopic analysis of an analyte as described in claim 30 whereinsaid diazonium compound sensor coating on said Raman spectroscopicsample surface establishes a covalent bond with a chemical on saidanalyte.
 33. A system for spectroscopic analysis of an analyte,comprising: a) Raman spectroscopic sample surface; b) at least one Ramanspectroscopic sample location on said spectroscopic surface; and c) anitroso compound sensor coating at said at least one spectroscopicsample location; wherein said analyte comprises nitroso-reactive analyteand wherein said nitroso compound sensor coating retains saidnitroso-reactive analyte.
 34. A system for spectroscopic analysis of ananalyte as described in claim 33, wherein said Raman spectroscopicsurface comprises a Surface Enhanced Raman Scattering surface.
 35. Asystem for spectroscopic analysis of an analyte as described in claim 33wherein said nitroso compound sensor coating on said spectroscopicsample surface establishes a covalent bond between said nitroso compoundsensor coating and said nitroso-reactive analyte.
 36. A system forspectroscopic analysis of an analyte, comprising: a) a Ramanspectroscopic sample surface, wherein said Raman spectroscopic samplesurface comprises a Surface Enhanced Raman Scattering surface; b) atleast one spectroscopic sample location on said Raman spectroscopicsurface; and c) an acyl compound sensor coating at said at least onespectroscopic sample location; wherein said analyte comprises anacyl-reactive analyte in a Raman spectroscopic sample, and wherein saidacyl compound sensor coating retains said acyl-specific analyte.
 37. Asystem for spectroscopic analysis of an analyte as described in claim 36wherein said acyl compound sensor coating on said spectroscopic samplesurface establishes a covalent bond with said acyl-reactive analyte. 38.A system for spectroscopic analysis of an analyte as described in claim36 wherein said acyl compound sensor coating on said Raman spectroscopicsample surface establishes a covalent bond between said acyl compoundsensor coating and an alcohol of said acyl-reactive analyte.
 39. Asystem for spectroscopic analysis of an analyte as described in claims30, 33 or 36, wherein said Raman spectroscopic sample surface furthercomprises a second compound sensor coating at said spectroscopic samplessurface location.
 40. A system for spectroscopic analysis of an analyteas described in claims 30, 33 or 36, wherein said compound sensorcoating further comprises: a) a Raman spectroscopic sample surfacecovalent attachment group; b) a molecular scaffolding attached to saidRaman spectroscopic sample surface attachment group; and c) a covalentbond between said molecular scaffolding and said compound sensorcoating.
 41. A system for spectroscopic analysis of an analyte asdescribed in claims 30, 33, or 36, and further comprising: a) a Ramanillumination device which illuminates said compound sensor coating onsaid Raman spectroscopic sample surface so as to produce a spectroscopicsignal; and b) a sensor positioned so as to receive said spectroscopicsignal.
 42. A method of spectroscopic analysis of an analyte, comprisingthe steps of: a) establishing a Raman spectroscopic sample surface; b)placing an analyte-reactive coating on said spectroscopic samplesurface; c) subjecting said analyte-reactive coating on saidspectroscopic sample surface to a sample; d) covalently bonding analytein said samples to said analyte-reactive coating on said Ramanspectroscopic sample surface; e) exposing said analyte-reactive coatingon said Raman spectroscopic sample surface to radiation having at leastone wavelength of energy; f) affecting a change in frequency of said atleast one wavelength of energy at least in part by said analytecovalently bonded to said analyte-reactive coating on said Ramanspectroscopic sample surface; and g) analyzing said change in frequencyof said at least one wavelength of energy to gain information about saidanalyte.
 43. A method of spectroscopic analysis of an analyte asdescribed in claim 42 further comprising the step of detecting asubstantially unaltered signal.
 44. A method of spectroscopic analysisof an analyte as described in claim 43 wherein said step of affecting achange in frequency of said at least one wavelength of energy at leastin part by said analyte covalently bonded to said analyte-reactivecoating on said Raman spectroscopic sample surface comprises Ramanscattering.
 45. A method of spectroscopic analysis of an analyte asdescribed in claim 42 wherein said step of placing an analyte-reactivesensor coating on said spectroscopic sample surface: a) establishing aRaman spectroscopic sample surface covalent attachment group; b)establishing a molecular scaffolding to said Raman spectroscopic surfacecovalent attachment group; and c) establishing a covalent attachmentbetween said molecular scaffolding and said analyte-reactive compoundsensor coating.
 46. A method of spectroscopic analysis of an analyte asdescribed in claim 42 further comprising the step of washing saidanalyte-reactive compound coating on said Raman spectroscopic samplesurface with a liquid.
 47. A method of spectroscopic analysis of ananalyte as described in claim 46 wherein said step of washing saidanalyte-reactive compound coating on said Raman spectroscopic samplesurface with a liquid removes at least one interfering substance fromsaid analyte-reactive compound coating on said Raman spectroscopicsample surface.
 48. A method for detection of a controlled substance asdescribed in claim 42 further comprising the step of affecting a changein amplitude of said at least one wavelength of energy at least in partby said analyte.
 49. A method for detection of a controlled substance asdescribed in claim 48 further comprising the step of analyzing saidchange in amplitude of said at least one wavelength of energy to gaininformation about said analyte.
 50. A Raman spectroscopic samplesurface, comprising: a) a Raman spectroscopic sample surface; b) ananalyte-reactive coating on said Raman spectroscopic sample surface; andc) a chemical reactive event location on said analyte-reactive coatingon said Raman spectroscopic sample surface to covalently bond an analyteto said analyte-reactive coating; and d) an internal standard, whereinsaid internal standard is co-located with said chemical reaction eventlocation.
 51. A system for spectroscopic analysis of an analyte asdescribed in claim 50, further comprising: a) an illumination devicewhich illuminates said chemical reaction event on said analyte-reactivecoating on said Raman spectroscopic sample surface so as to produce aRaman spectroscopic signal; and b) a sensor positioned so as to receivesaid Raman spectroscopic signal.
 52. A system for spectroscopic analysisof an analyte as described in claim 50 further comprising a) a Ramanspectroscopic surface attachment group; b) a molecular scaffoldingattached to said Raman spectroscopic surface attachment group; and c) acovalent bond between said molecular scaffolding and saidanalyte-reactive coating.
 53. A system for spectroscopic analysis of ananalyte as described in claim 52 wherein said analyte-reactive coatingcomprises a precursor reactive functional group attached to said centralmolecular scaffolding, wherein said precursor reactive functional groupwhen activated becomes said chemical reaction event location.
 54. Asystem for spectroscopic analysis of an analyte, comprising: a) a Ramanspectroscopic sample surface; b) an analyte-reactive coating attached tosaid Raman spectroscopic sample surface comprising: 1) a Ramanspectroscopic sample surface attachment group; 2) a central molecularscaffolding attached to said surface attachment group; and 3) a reactivefunctional group attached to said central molecular scaffolding whereinsaid reactive functional group is adapted to react with an analyte toform a covalent bond; and c) an internal standard, wherein said internalstandard is co-located with said chemical reaction event location.
 55. Asystem for spectroscopic analysis of an analyte as described in claim 54wherein said central molecular scaffolding attached to said surfaceattachment group is selected from the group consisting of a monocyclicaromatic ring, a polycyclic aromatic ring, a heteroaromatic ring, and anon-aromatic scaffolding.
 56. A system for spectroscopic analysis of ananalyte as described in claim 54 wherein said reactive functional groupattached to said central molecular scaffolding is selected from thegroup consisting of a diazonium group, a diazonium salt, a nitroso, anactivated acyl sensor, an electrophilic carbon, and a carbonyl.
 57. Asystem for spectroscopic analysis of an analyte, comprising: a) a Ramanspectroscopic sample surface; b) an analyte-reactive coating on saidRaman spectroscopic sample surface, comprising: 1) a Raman spectroscopicsample surface attachment group; 2) a central molecular scaffoldingattached to said surface attachment group; and 3) a reactive functionalgroup precursor attached to said central molecular scaffolding whereinsaid reactive functional group is adapted to be activated to become areactive functional group attached to said central molecular scaffoldingand wherein said reactive functional group attached to said centralmolecular scaffolding reacts with said analyte to form a covalent bond;and c) an internal standard, wherein said internal standard isco-located with said chemical reaction event location.
 58. A system forspectroscopic analysis of an analyte as described in claim 57 furthercomprising an activation substance which activates said reactivefunctional group precursor to become said reactive functional group. 59.A system for spectroscopic analysis of an analyte as described in claim58 wherein said reactive functional group precursor forms an adduct withsaid analyte after its activation by said activation substance.
 60. Asystem for spectroscopic analysis of an analyte as described in claims54 or 57 wherein said internal standard comprises a tether.
 61. A systemfor spectroscopic analysis of an analyte as described in claims 54 or 57wherein said surface attachment group and said central molecularscaffolding form at least part of a coating, and wherein said internalstandard is selected from the group consisting of a non-carbonstructure, a nitro group, a sulfonyl-group, a carbon structure, and acarbonyl group.
 62. A method of spectroscopic analysis of an analyte,comprising the steps of: a) establishing a first sensor molecule type ona Raman spectroscopic sample surface; b) establishing a second sensormolecule type on said Raman spectroscopic sample surface; c) subjectingsaid spectroscopic sample surface to an analyte; d) covalently bondingsaid analyte to said first sensor molecule type; e) interacting saidanalyte with said second sensor molecule type; f) exposing saidspectroscopic sample surface to radiation having at least one wavelengthof energy; g) affecting a change in frequency of said at least onewavelength of energy at least in part by said analyte; and h) analyzingsaid change in frequency of said at least one wavelength of energy togain information about said analyte.
 63. A method of spectroscopicanalysis of an analyte as described in claim 62 wherein said step ofinteracting said analyte with said second molecule type comprises thestep of forming a covalent bond between said analyte and said secondsensor molecule type.
 64. A method of spectroscopic analysis of ananalyte as described in claim 62 further comprising the step ofaffecting a change in amplitude of said at least one wavelength ofenergy at least in part by said analyte.
 65. A method of spectroscopicanalysis of an analyte as described in claim 64 further comprising thestep of analyzing said change in amplitude of said at least onewavelength of energy to gain information about said analyte.
 66. Amethod of spectroscopic analysis of an analyte, comprising the steps of:a) establishing a first sensor molecule type on a Raman spectroscopicsample surface; b) establishing a second sensor molecule type on saidRaman spectroscopic sample surface; c) subjecting said Ramanspectroscopic sample surface having said first sensor molecule type andsaid second sensor molecule type to samples at least a portion of whichcontain an analyte; d) interacting said analyte with said first sensormolecule type; e) interacting said analyte with said second sensormolecule type; f) exposing said spectroscopic sample surface toradiation having at least one wavelength of energy; g) affecting achange in frequency of said radiation having at least one wavelength ofenergy at least in part by said analyte bound to said first sensormolecule type or to said second sensor molecule type; and h) analyzingsaid change in frequency of said at least one wavelength of energy togain information about said analyte.
 67. A method of spectroscopicanalysis of an analyte as described in claim 66 wherein said Ramanspectroscopic sample surface comprises a Surface Enhanced RamanScattering spectroscopic sample surface.
 68. A method of spectroscopicanalysis of an analyte as described in claim 66 further comprising thestep of affecting a change in amplitude of said at least one wavelengthof energy at least in part by said analyte bound to said first sensormolecule type or to said second sensor molecule type.
 69. A method ofspectroscopic analysis of an analyte as described in claim 68 furthercomprising the step of analyzing said change in amplitude of said atleast one wavelength of energy to gain information about said analyte.70. A method of spectroscopic analysis of an analyte, comprising thesteps of: a) establishing a first sensor molecule type on a Ramanspectroscopic sample surface which responds to a first concentrationrange of said analyte; b) establishing a second sensor molecule type onsaid Raman spectroscopic sample surface which responds to a secondconcentration range of said analyte wherein said first concentrationrange and said second concentration range are different; c) subjectingsaid Raman spectroscopic sample surface to an analyte; d) interactingsaid analyte with said first sensor molecule type; e) interacting saidanalyte with said second sensor molecule type; f) exposing said Ramanspectroscopic sample surface to radiation having at least one wavelengthof energy; g) affecting frequency of said radiation having at least onewavelength of energy at least in part by said analyte; and h) analyzingchange in frequency of said radiation having at least one wavelength ofenergy to gain information about said analyte.
 71. A method ofspectroscopic analysis of an analyte as described in claim 70, whereinsaid first concentration range and said second concentration range havea concentration range overlap region created by said first sensormolecule type and said second sensor molecule type.
 72. A method ofspectroscopic analysis of an analyte as described in claim 71 whereinsaid first sensor molecule type has a first saturation level for saidreactive analyte, wherein said second sensor molecule type has a secondsaturation level for said reactive analyte, and further comprising thestep of establishing different saturation levels for said first sensormolecule type and said second sensor molecule type.
 73. A method ofspectroscopic analysis of an analyte as described in claim 72 whereinsaid step of establishing different saturation levels for said firstsensor molecule type and said second sensor molecule types comprises thestep of establishing complementary ranges provided by said first sensormolecule type and said second sensor molecule type.
 74. A method ofspectroscopic analysis of an analyte as described in claim 70 furthercomprising the step of affecting a change in amplitude of said at leastone wavelength of energy at least in part by said analyte.
 75. A methodof spectroscopic analysis of an analyte as described in claim 74 furthercomprising the step of analyzing said change in amplitude of said atleast one wavelength of energy to gain information about said analyte.76. A method of spectroscopic analysis of an analyte as described inclaim 62 or 70 and further comprising the steps of: a) establishing afirst modifier attached to said first sensor molecule type on saidspectroscopic sample surface; and b) establishing a second modifierattached to said second sensor molecule type on said spectroscopicsample surface.
 77. A method of spectroscopic analysis of an analyte asdescribed in claim 76 wherein said first modifier and a second modifierare selected from the group consisting of a halide, an electron-donatinggroup, an alkoxy group, amide group, amino group, anelectron-withdrawing group, a nitro group, a carboxylic acid group, anester group, a sulfoxide group, and a sulfone.
 78. A method forspectroscopic analysis of multiple species of analytes, comprising thesteps of: a) establishing a first sensor molecule type on a Ramanspectroscopic sample surface; b) establishing at least a second sensormolecule type on said Raman spectroscopic sample surface; c) subjectingsaid spectroscopic sample surface to at least a first species of analytewherein said first species of analyte reacts with said first sensormolecule type on said spectroscopic sample surface; d) subjecting saidRaman spectroscopic sample surface to at least a second species ofanalyte wherein said second species of analyte reacts with said secondsensor molecule type on said spectroscopic sample surface; e) covalentlybonding said first species of analyte to said first sensor moleculetype; f) interacting said second species of analyte with said secondsensor molecule type; g) exposing said Raman spectroscopic samplesurface to radiation having at least one wavelength of energy; h)affecting frequency of said radiation having at least one wavelength ofenergy at least in part by said first species of analyte; i) affectingsaid frequency of radiation having at least one wavelength of energy atleast in part by said second species of analyte; and j) simultaneouslyanalyzing change in frequency of said radiation having at least onewavelength of energy due to said second species of analyte to gaininformation about said second species of said analyte.
 79. A method forspectroscopic analysis of multiple species of analytes as described inclaim 78 wherein said step of covalently bonding said first species ofsaid analyte to said first sensor molecule type comprises the step offorming a first adduct type between said first species of said analyteand said first sensor molecule type.
 80. A method for spectroscopicanalysis of multiple species of analytes as described in claim 78wherein said step of covalently bonding said first species of saidanalyte to said first sensor molecule type comprises the step of forminga first adduct type between said first species of said analyte and saidfirst sensor molecule type, and wherein said step of interacting saidsecond species of said analyte with said second sensor molecule typecomprises the step of forming a second adduct type between said secondspecies of said analyte and said second sensor molecule type.
 81. Amethod for spectroscopic analysis of multiple species of analytes asdescribed in claim 78 wherein said step of simultaneously analyzing saidchange in frequency of said radiation having at least one wavelength ofenergy to gain information about said second species of analytecomprises the step of differentiating information between said firstspecies of analyte and said second species of said analyte.
 82. A methodfor spectroscopic analysis of multiple species of analytes as describedin claim 81 wherein said step of differentiating information betweensaid first species of analyte and said second species of analytecomprises the step of quantitating said first species of analyte andquantitating said second species of analyte.
 83. A method ofspectroscopic analysis of an analyte as described in claims 78, 81, or82 further comprising the step of affecting a change in amplitude ofsaid at least one wavelength of energy at least in part by said secondspecies of analyte.
 84. A method of spectroscopic analysis of an analyteas described in claim 83 further comprising the step of analyzing saidchange in amplitude of said at least one wavelength of energy to gaininformation about said second species of analyte.
 85. A system forspectroscopic analysis of an analyte, comprising: a) Raman spectroscopicsample surface; b) a first sensor molecule type on said Ramanspectroscopic sample surface that forms a covalent bond with an analytesubjected to said Raman spectroscopic sample surface; and c) a secondsensor molecule type on said Raman spectroscopic sample surface.
 86. Asystem for spectroscopic analysis of an analyte as described in claim 85wherein said second sensor molecule type forms a covalent bond betweenitself and said analyte subjected to said Raman spectroscopic samplesurface.
 87. A system for spectroscopic analysis of an analyte asdescribed in claim 85 wherein said Raman spectroscopic sample surfacecomprises a Surface Enhanced Raman Scattering surface.
 88. A system forspectroscopic analysis of an analyte, comprising: a) a Ramanspectroscopic sample surface; b) a first sensor molecule type on saidRaman spectroscopic sample surface which has a first concentration rangefor an analyte; and c) a second sensor molecule type on said Ramanspectroscopic sample surface which has a second concentration range forsaid analyte wherein said first concentration range and said secondconcentration range are different.
 89. A system for spectroscopicanalysis of an analyte as described in claim 88 wherein said firstconcentration range and said second concentration range of said firstsensor molecule type and said second sensor molecule type on said Ramanspectroscopic sample surface have an overlap region.
 90. A system forspectroscopic analysis of an analyte as described in claim 89 whereinsaid first sensor molecule type has a first saturation level for saidanalyte, and wherein said second sensor molecule type has a secondsaturation level for said analyte, and wherein said first saturationlevel and second saturation level are different.
 91. A system forspectroscopic analysis of an analyte, comprising: a) Raman spectroscopicsample surface; b) a first sensor molecule type on said Ramanspectroscopic sample surface which has a first analytical range; and c)a second sensor molecule type on said Raman spectroscopic sample surfacewhich has a second analytical range, and wherein said first analyticalrange and said second analytical range are complementary.
 92. A systemfor spectroscopic analysis of an analyte, comprising: a) a Ramanspectroscopic sample surface; b) a first sensor molecule type on saidRaman spectroscopic sample surface; c) a first modifier attached to saidfirst sensor molecule type that influences spectral characteristics ofradiation having at least one wavelength; d) a second sensor moleculetype on said Raman spectroscopic sample surface; and e) a secondmodifier attached to said second sensor molecule type on said Ramanspectroscopic sample surface.
 93. A system for spectroscopic analysis ofan analyte, comprising: a) a Raman spectroscopic sample surface; b) afirst sensor molecule type on said Raman spectroscopic sample surfacewherein said first sensor molecule type covalently bonds to a first typeof analyte; and c) at least a second sensor molecule type on said Ramanspectroscopic sample surface wherein said second sensor molecule typeresponds to a second type of analyte, and wherein said first type ofanalyte and said second type of analyte are different.
 94. A system forspectroscopic analysis of an analyte as described in claim 93 whereinsaid first sensor molecule type on said Raman spectroscopic samplesurface covalently bonds to said first type of analyte to form a firsttype of adduct for spectroscopic analysis.
 95. A system forspectroscopic analysis of an analyte as described in claim 93 whereinsaid second sensor molecule type on said Raman spectroscopic samplesurface covalently bonds to said second type of analyte to form a secondtype of adduct for spectroscopic analysis.